Necroptosis, a programmed and regulated form of necrosis, has emerged as an important mechanism in the pathogenesis of various forms of lung injuries resulting from respiratory virus infection, trauma, blood transfusion, chronic obstructive pulmonary disease, and sepsis. Unlike apoptosis, necroptosis causes cell membrane rupture and triggers inflammation through the release of damage-associated molecular patterns, such as mitochondrial DNA, high-mobility group protein B1, and interleukin-1. Necroptosis can be induced by the activation of tumor-necrosis factor (TNF) receptor, Toll-like receptors (TLR) 3 and 4 and by virus infection. Necroptosis induced by TNF receptor and TLR3 contributes to host cell death by respiratory virus infection as infection of both DNA and RNA viruses normally induces the production of TNF and double-stranded RNA (dsRNA). The current prevailing model of necroptosis is executed by receptor-interacting protein kinase-3 (RIPK3) and the RIPK3-mediated phosphorylation of mixed lineage kinase domain-like protein (MLKL). The central RIPK3-MLKL necroptosis pathway was originally discovered in transformed cancer cell lines and studied in murine models. In contrast, little has been done for necroptosis signaling pathway in primary normal human cells. This issue is considered important and should be addressed before moving the currently identified necroptosis targets to clinical trials. Hence, we seek to identify and define the necroptosis signal pathways in primary normal human lung epithelial cells. On the basis of our preliminary data, this resubmission R21 proposal will test the hypothesis that normal human lung epithelial cells may undergo necroptosis through novel signal pathways mediated by a yet unidentified RIPK1-independent but necrostatin-1/1s-sensitive kinase, which is different from murine lung epithelial cells. We will test the novel hypothesis in two Specific Aims. Aim 1 will identify the novel necrostatin-1/1s-sensitive kinase in human alveolar epithelial cells by utilizing human kinase siRNA library screening and if needed, an alternative proteomic approach; Aim 2 will define the novel kinase-mediated necroptosis signal pathways by TNF and dsRNA in primary human alveolar and airway epithelial cells. We anticipate that this proposal will identify the novel necrostatin-1/1s-sensitive kinase responsible for necroptosis in primary human lung epithelial cells. These studies will address gaps in our understanding of necroptosis signal pathways in primary normal human cells and thereby advance the field. The proposal may also identify a promising novel therapeutic target that could be further developed to control various forms of human lung injuries, in which lung epithelial cell death is generally a prominent feature.